Apoptosis has recently been attracting attention with regard to the death of cells or tissues. Unlike pathologic death of cells (necrosis), apoptosis is considered to be the death programmed in the gene. of cells from the beginning. That is, certain external or internal factors trigger to activate the genes which program apoptosis and the active death is brought to the cell as a result of the activation of this self-destructive program.
TABLE 1 ______________________________________ Phenomena of apoptosis ______________________________________ [Physiological phenomena] developmental stage morphogenesis, metamorphosis, establishment of nervous system alternation of normal hemocyte, epidermal cells, epithelial cells cells of small intestine and stomach nervous system death of neurons due to the removal of neurotrophic factors endocrine death of thymocytes by glucocorticoid atrophy of prostate by androgen removal immune system death of autoimmune cells death of tumor cells by cytotoxic T lymphocytes [Pathological phenomena] irradiation death of thymocytes highly sensitive to irradiation viral infection cell death by infection with AIDS or influenza virus cancer death of tumor cells in malignant tissues drug, poison cell death by antitumor agents or bacterial toxins heat death of tumor cells by thermotherapy ______________________________________
As shown in Table 1 above, apoptosis is involved in a great number of vital phenomena. It is suggested that apoptosis is responsible for not only morphogenesis during developmental stages, but also alternation of normal cells (removal of old cells), such as epidermal cells of skin and epithelial cells of small intestine and stomach of mature individuals, atrophy of hormone-dependent tissue thymus by glucocorticoid, atrophy of prostate by castration, elimination of immunocompetent cells which react with self-components, and death of neurons due to the removal of neurotrophic factors.
In addition to such physiological death of cells, apoptosis is also found in the death of cells exposed to irradiation and virus-infected cells. A decrease in T lymphocytes due to AIDS virus has been reported to be also caused by apoptosis, which attracted much attention. Besides these, apoptosis is caused by chemical or physical stimulation, such as administration of medications and poisonous substances, and heat. The death of neurons in neurodegenerative diseases such as Alzheimer's disease, and natural apoptosis of tumor cells and cell death by antitumor agents which occur in malignant tumor lesion have been found to be caused by apoptosis.
Thus, elucidation of molecular mechanisms of apoptosis is crucial for the understanding of biochemical significance and role of cell death in ontogenesis and suppression of carcinogenesis.
The characteristic phenomena commonly observed in apoptosis are morphological changes such as contraction of cell along with the changes in cell membrane (elimination of microbilli) and condensation of chromatin, and fragmentation of chromatin DNA. [Br. J Cancer 26, 239-257 (1972), Nature 284, 555-556 (1980)]. In particular, fragmentation of chromatin DNA into nucleosomal units (FIG. 1) is the most noticeable phenomenon commonly seen in every apoptotic cell, irrespective of the diversity of causative factors of apoptosis, which suggests that apoptosis cascade ends in the process of fragmentation of chromatin DNA.
It has been conventionally suggested that the cleavage of chromatin DNA found in apoptosis is catalyzed by a Zn.sup.2+ sensitive, endogenous Ca.sup.2+ dependent endonuclease [J. Immunol. 132, 38-42 (1984), J. Biol. Chem. 266, 18580-18585 (1991), EMBO J. 12, 371-377 (1993), Biochemistry 32, 9129-9136 (1993)].
Motivated by the suggestion, several kinds of endonucleases (Nuc 118, DNase I, DNase II) considered to be the enzymes possibly involved in apoptosis have been recently purified from thymus or cultured cells [Biochem. Biophys. Res. Commun. 39, 254-259 (1970), J. Biol. Chem. 266, 18580-18585 (1991), EMBO J. 12, 371-377 (1993), Arch. Biochem. Biophys. 300, 440-450 (1993)].
However, such enzymes have been purified from normal cells and there is no conclusive proof to show actual involvement of such enzymes in apoptosis.
Therefore, elucidation of endonuclease involved in the fragmentation of chromatin DNA in apoptosis and the control mechanism thereof is extremely important for understanding the whole picture of molecular mechanism of apoptosis, as well as the mechanism of viability and death of cells. It is also useful for developing an agent for the diagnosis, prevention or therapy of cancer, autoimmune diseases, AIDS and the like, in which apoptosis plays certain roles.